Gene therapy, which refers to treating diseases by delivering nucleic acids for treatment to desired organs in the body and expressing new proteins in cells, is a method that does not treat symptoms of diseases but treats the causes of diseases and removing the diseases. Gene therapy can have excellent selectivity compared to the general treatments by drugs, and can be applied for a long time by improving cure rates and treatment pace of the diseases that are difficult to be controlled by other treatment methods. As nucleic acids for treatment, DNA is vulnerable to hydrolysis caused by in vivo enzymes and has low efficiency in entering the cells, and therefore, it is necessary to develop a gene carrier that can safely deliver nucleic acids to desired target cells to achieve high expression efficiency for an effective gene therapy.
A gene transporter should have low or no toxicity and be able to deliver genes to desired cells selectively and effectively. These gene carriers are largely classified into viral and non-viral ones. Until recently, for clinical trials, viral vectors that have high transfection efficiency have been used as a gene carrier. However, viral vectors, such as retrovirus, adenovirus, and adeno-associated virus, not only have complex preparation steps, but also have safety problems, such as immunogenicity, infection risk, induction of inflammation, insertion of non-specific DNA, etc., and the problem in that the acceptable DNA size is limited. Therefore, the viral vectors have limitations to be applied in the body. As such, at present, non-viral vectors have gained attention as a replacement for viral vectors.
Non-viral vectors have advantages, such as repeated administrations with minimal immune response, enabling specific delivery to particular cells, excellent storage stability, and easy mass production. Examples of these non-viral vectors may include cationic liposomes, such as N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethyl ammonium chloride (DOTMA), alkylammonium, cationic cholesterol derivatives, gramicidin, etc.
Lately, there has been increasing attention since cationic polymers among non-viral vectors can form a complex through an ionic bond with anionic DNA. Such cationic polymers include poly-L-lysine (PLL), poly (4-hydroxy-L-proline ester), polyethylenimine (PEI), poly-[α-(4-aminobutyl)-L-glycolic acid], polyamidoamine dendrimer, poly-[N,N′-(dimethylamino)-ethyl]-methacrylate (PDMAEMA), etc., and these polymers compress DNA and form nanoparticles to protect DNA from degradation by enzymes and help to invade into cells rapidly and escape from endosomes. Most non-viral vectors have advantages, such as biodegradability, low toxicity, non-immunogenicity, convenience for use, etc., but have problems, such as relatively low transfection efficiency, limited particle size, etc.
Specifically, although most cationic polymers used as non-viral vectors show high transfection efficiency in vitro, which is an environment with low blood serum concentration, the transfection efficiency of the cationic polymer/gene complex is considerably inhibited by various factors present in the blood serum in vivo, thereby making gene entry into cells difficult. This is because non-specific interactions with plasma proteins and blood compositions are induced by an excessive positive electric charge caused on the surface of the cationic polymer/gene complex in vivo. Therefore, the transfection efficiency of cationic polymers is considerably decreased in vivo where a lot of blood serum is present, but not in vitro, where serum-free media or very low concentration of blood serum is present. When this is applied in vivo as it is, aggregates and accumulations in the lung, liver, and spleen, and furthermore opsonization and removal by reticuloendothelial system may be caused. Therefore, it is inevitable to limit the medical applications of the cationic polymers. The PEI, which has been most broadly studied as a non-viral vector, also has considerably low in vivo transfection efficiency and has a problem such as high cytotoxicity and low effects on gene expression due to low blood compatibility. Therefore, there is a need for developing the gene carrier that can enhance transfection efficiency while maintaining the advantages of the existing non-viral vectors.
In particular, the most challenging task in increasing the cure rate by gene therapy was on how to increase the delivery rate of the nucleic acids for treatment that cross biological barriers such as cell membranes, tumor tissues, and the blood brain barrier (BBB). Although various gene therapy targets have been discovered due to recent research on brain diseases, the effects are difficult to be proven because of a lack of means to effectively apply the gene therapy targets to animal models.
Specifically, the BBB, which is a cerebrovascular structure that limits the delivery of substances from the blood to brain tissues, is known to be formed mostly by tight junctions of cerebral capillary endothelium, known to surround blood vessels, and have impermeability to giant molecules such as nucleic acids. Particularly, fat-soluble substances are known to traverse the BBB, but non fat-soluble substances including polar substances, strong electrolytes, etc., are not really known to transmigrate the BBB. Although there is an advantage in that the brain tissues are protected from harmful substances by the BBB, there is a disadvantage in that the accessibility to treatment substances is decreased compared to other tissues in the body by blocking the delivery of radioisotopes, dyes, drugs, etc., required for the treatment of the brain tissues. Under the circumstance where even the delivery of polar compounds to brain tissues through the BBB is not easy, the delivery of nucleic acids, which are large molecules with strong polarity, is even more difficult. Besides the BBB, biologically-hindering mechanisms, such as decomposition by nuclease, immune clearance, difficulty in cell influx, off-target deposition in vivo, etc., make the gene delivery to the brain tissues difficult. Therefore, there is a need for developing a gene transporter that can overcome the hindering mechanisms and perform effective gene therapy for the brain tissues.
Because the gene delivery using most viral vectors is not able to cross the BBB by systemic delivery, direct injection/insertion into the brain is generally performed. However, there are problems where transfection has limited insertion sites and direct injection method is non-invasive for brain tissues. As such, in order to increase the delivery efficiency of substances to brain tissues by systemic delivery, there has been an attempt to increase permeability of the BBB by an intra-arterial injection of an osmotic agent such as mannitol. Specifically, the tissues were pretreated with hyperosmotic mannitol to loosen the tight junctions between the cells, followed by the treatment of various gene/drug delivery vehicles. However, the effect of mannitol was temporary and disappeared after 30 minutes, and the effect disappeared even before the influx of drugs or DNA. Further, the systemic delivery of mannitol has brought an effect of an overall increase of permeability to the BBB and thus it was not possible to specifically increase the permeability of particular substances for delivery.
Even if the genes have transmigrated the BBB, they still have to safely go through with cellular uptake and endosomal trapping to be transported to the cells. Therefore, the procedure for delivering genes to target cells of tissues is the biggest obstacle and technical problem to be solved, in terms of gene therapy for animals.
The present inventors have developed novel gene transporters, and suggested transporters capable of binding to nucleic acids based on mannitol and sorbitol and delivering the nucleic acids into the cells. However, in the field of gene transporters, there is yet a continuous demand for developing gene transporters that have higher delivery efficiency and can effectively deliver the nucleic acids to specific tissues.